The 174-base prohead RNA (pRNA) of bacteriophage o29 is an essential component of the molecular motor that packages the viral genomic DNA into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating forces greater than 60 pN. o29 is a premier model system for DNA packaging and serves as a model for analogous processes in certain animal viruses, such as herpes- and adenoviruses. pRNA binds to the prohead by forming a novel cyclic pentamer via intermolecular base pairing between identical molecules. The RNA ring then provides a scaffold upon which the packaging ATPase assembles into its functional ring shape. In addition to providing a scaffold function, pRNA is hypothesized to function in higher order processes of packaging, such as coordination and communication within the motor. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance in uncovering targets for antiviral agents. Additionally, the properties of pRNA are being exploited for nanomedicine applications. The ultimate goal of the research is to determine the structure and functional roles of pRNA in the mechanism of DNA translocation. Here we employ a highly integrated, multi-disciplinary approach, including genetic and biochemical analysis, X-ray crystallography, cryoEM 3-D reconstruction and single molecule laser tweezers to investigate the structure/function relationship of pRNA: 1) X-ray crystallography will be used to complete the atomic structure of pRNA, and pRNA- ATPase complex formation will be characterized by biochemical analysis; 2) characterize functional elements of pRNA involved in motor assembly, communication and coordination by site-directed mutagenesis and chimeric RNAs; and 3) investigate pRNA-mediated assembly and motor communication and coordination using ordered heteromeric pRNA rings containing wild-type and mutant pRNAs.